Killer toxins (KTs) are proteins secreted by yeasts which are able to kill other yeasts or microorganisms which compete in nature for the same ecological niche [1]. Although they are attractive therapeutic tools, due to their wide spectrum of microbicidal activity, they are of no practical use because of their instability in the host physiological milieu as well as their antigenicity and toxicity. Instead, the use of anti-idiotypic antibodies which mimic KTs has been shown to be effective.
The killer toxin from Pichia anomala (‘PaKT’) has a wide spectrum of microbicidal activity against pathogens including Candida albicans, Aspergillus fumigatus, Pneumocystis carinii, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus [2, 3, 4]. This observation has been exploited by the generation of a PaKT-neutralizing monoclonal antibody in mice (mAb KT4) [5] whose idiotype (Id) is able to induce the production of anti-idiotypic antibodies (antilds) [6, 7, 8]. These antilds represent the internal image of the active PaKT domain and as such exert its biological activities, including binding to the PaKT receptor (KTR) of susceptible microorganisms and broad spectrum microbicidal activity overlapping that of PaKT (FIG. 1).
Experimental animals in which these antibodies (′KTIdAb ) are raised by idiotypic vaccination with mAb KT4 have repeatedly been shown to be protected against mucosal or systemic challenges by C.albicans [7, 8]. There is also ample evidence of susceptibility in vitro to KTIdAb by diverse microbial pathogens such as M.tuberculosis (including multidrug resistant strains), P.carinii, and others [4, 9].
Idiotypic theory (FIG. 1) also predicted that antibodies against PaKT receptors would mimic PaKT activity. This has been demonstrated in animals and humans during the course of experimental and natural infections caused by PaKT-sensitive microorganisms [10]. Human natural anti-KTR antibodies have been shown to have microbicidal activity in vitro against C.albicans, M.tuberuclosis, and P.carinii, to inhibit P.carinii infectivity of nude rats, and to be protective against passive transfer in vivo, in an experimental model of rat vaginal candidiasis [4, 10, 11].
Based on these results, and in order to obtain standard KTldAb in sufficient amounts, rat monoclonal IgM (mAb K10) and mouse single-chain Fv (scFv H6) microbicidal antibodies have been obtained [12, 13]. These two antibodies have strong microbicidal effects in vitro against important pathogenic microorganisms including: C.albicans [12, 13]; C.krusei and C.glabrata (including fluconazole-resistant strains); Cryptococcus neoformans; A. fumigalus [14]; M.tuberculosis [4]; S.aureus, Enterococcus faecalis, E.faecium, and Streptococcus pneumoniae (including methicillin-, vancomycin- and penicillin-resistant strains) [15], S.mutans, Leishmania major, L.infantum and Achantamoeba castellani. Furthermore, they showed specific therapeutic activity in an experimental model of rat vaginal candidiasis by intravaginal administration [13]. In addition, K10 proved to be therapeutic against P.carinii pneumonia in rats infected by aerosol administration [16], and in mice transplanted with T cell depleted bone marrow against aspergillosis caused by nasal instillation [14].
Although the existence of scFv H6 has been reported, a method for its manufacture has not previously been disclosed, and nor has its amino acid sequence.
It is an object of the invention to provide further and improved antimicrobial and/or antiviral compounds.